The Role Of WboA Gene In Brucella S₂Strain Inducing Immunity

ObjectiveTo explore the role of WboA gene encoding products inducing immunity againstinfection, and provide the basis for the feasibility of the development of Brucella vaccineswithout WboA.Methods1.The roles of WboA gene encoding products druing Brucella infecting macrophages anddendritic cells:Macrophages and dendritic cells were respectively infected by Brucella suis strainsWboA-S2(MOI100:1), S2strain as a control, in order to observe their characteristics such asthe histological changes (1h,12h,24h), phagocytosis, the microbial load(1h,6h,12h,24h),apoptosis and the change of cytokines.2. The roles of WboA-S2strains during stimulating BMDC and MΦ to present antigen:BMDC and MΦ infected by WboA-S2strains were respectively co-cultured with T cells,S2strain as a control, in order to observe the changes of IFN-γ and IL-4levels(24h,48h,72h)in culture at different time points and identify the roles of WboA gene on presenting antigenand activating T cell responses by BMDC and MΦ.Results1. The histological changes of MΦ and BMDC infected by WboA-S2strains and S2strainsThe results were observed by Wright-Giemsa staining under an optical microscope: therewere a lot of increased vacuoles and brucellosis in MΦ infected1h, and the completement ofmembrane of macrophages infected by WboA-S2strains was better than that of S2strains;there were still a lot of brucellosis in MΦ infected by them12h, and the membrane ofmacrophages was still complete; At24h, the membrane of macrophages infected by S2disappeared and the nuclear was condensate while the membrane of macrophages infected byWboA-S2strains was more complete; BMDC infected by Brucella did not changesignificantly: at1h the volume of BMDC was slightly increased, there were only a small number of bacteria, cell cytoplasm more complete, only a few cell membrane damage at24h.2. The phagocytic rate of MΦ and BMDC infected by WboA-S2strains and S2strainsInfected by S2strains at1h, the phagocytic rate of MΦ was (43.6±4.8)%, which weresignificantly higher than those of BMDC (13.08±2.36)%(P<0.05); Infected by WboA-S2strains at1h, the phagocytic rates of MΦ and BMDC were both higher than that of S2(P<0.05). It was shown that WboA-S2strains was more killed and phagocytosed by MΦ andBMDC.3. The changes of the microbial load in MΦ and BMDC infected by WboA-S2strains and S2strains at different time pointsAfter infected by WboA-S2strains at1h, the microbial load in MΦ and BMDC were bothhigher than that of S2strains. With the extension of the interaction time, the microbial load ofeach group declined. After infected by WboA-S2strains at24h,the microbial load of cells wassignificantly lower than that of S2(P <0.01). It was shown that WboA-S2strains was morekilled and cleared away by MΦ and BMDC.4. The comparison of IL-12and TNF-α secreted by macrophages and dendritic cellsinfected by WboA-S2strainsMΦ groups:It was shown that IL-12and TNF-α in cocultivation supernatant secreted bymacrophages infected by WboA-S2strains and S2strains were all higher than those of normalcontrol group at each time point (P<0.01).It was illustrated that WboA-S2strains and S2trainscould activate MΦ, and induced differentiation of Th0to Th1,stimulating cellular immuneresponse; IL-12and TNF-α secreted by macrophages infected by WboA-S2strains was bothsignificantly higher than that of S2strains (P<0.01). It was illustrated that MΦ was moresensitive to the stimulation of WboA-S2strains.BMDC groups: It was shown that TNF-α and IL-12in cocultivation supernatant secretedby dendritic cells infected by WboA-S2strains and S2strains were all higher than those ofnormal control group at each time point (P<0.01), and IL-12and TNF-α secreted by dendriticcells infected by WboA-S2strains was both significantly higher than that of S2strains (P<0.05).It was illustrated that WboA-S2strains and S2trains could also activate dendritic cells, and stimulate the body to produce cellular immune response; And BMDC was more sensitive toWboA-S2strains than S2strains.5. Detecting apoptosis rate of MΦ and BMDC infected by WboA-S2strains and S2strainsThe results showed that the apoptosis rate of MΦ infected by WboA-S2was dramaticllyhigher than that of S2group and normal group (P<0.05), while there was not statisticallysignificant between the apoptosis rates of BMDC infected by WboA-S2or S2and that ofnormal BMDC.6. The secretion of IFN-γ in cocultivation supernatant from macrophages trained with T Cellsafter infected by WboA-S2and S2The results showed that compared with the control group, IFN-γ from na ve T Cellsstimulated by MΦ infected by WboA-S2and S2was higher than normal group (P<0.05),while IFN-γ from WboA-S2group was higher than that of S2group(P<0.01).It was illustratedthat WboA-S2and S2could indruce MΦ to present antigen in order to produce cellular immuneresponse, the ability of WboA-S2to stimulate MΦ to present antigen was much stronger thanthat of S2. The results for the first time proved MΦ may be offered Brucella antigen, activateinitial T cells, and the capability of MΦ that loaded with WboA-S2strains antigen to startinitial T cell responses was more stronger.7. The secretion of IFN-γ in cocultivation supernatant from BMDC trained with T Cellsafter infected by WboA-S2and S2The results showed that compared with the control group, IFN-γ from T Cellsstimulated by BMDC infected by WboA-S2and S2was higher than normal group (P<0.01),and IFN-γ from WboA-S2group was much higher than that of S2group (P<0.01).It wasillustrated that WboA-S2and S2could indruce BMDC to present antigen in order to producecellular immune response, the ability of WboA-S2to stimulate BMDC to present antigen wasmuch stronger than that of S2.8. The secretion of IL-4in cocultivation supernatant from MΦ and BMDC trained with TCells after infected by WboA-S2and S2The results showed that the secretion of IL-4from MΦ and BMDC were not detected during WboA-S2group, S2group and normal group.It was illustrated that MΦ and BMDCinfected by WboA-S2and S2did not stimulate Th0to Th2,and did not produce humoralimmune response.Conclusion1. Compared with S2strains, WboA-S2strains is more susceptible to MΦ and BMDCphagocytosis and clearance, and the ability of WboA-S2strains to induce apoptosis is stronger.2. The ability of WboA-S2strains to activate MΦ and BMDC is more powerful than that of S2strains, and the ability of MΦ and BMDC infected by WboA-S2strains to present antigen andinduce T cell activation is higher than that of S2strains. The WboA-S2strains as Brucellavaccine can induce stronger immune response to Brucella.